Characterization of mAb size heterogeneity originating from a cysteine to tyrosine substitution using denaturing and native LC-MS

Upon assessing the comparability between a biosimilar mAb and its reference product by non-reducing CE-SDS, increased levels of a heavy-heavy-light chain (HHL) variant, present as a low molecular weight (LMW) peak, were observed. RPLC-MS applied at top, middle-up and bottom-up level revealed the existence of Cys-to-Tyr substitutions, predominantly at position HC226 involved in connecting LC and HC, explaining the abundant HHL levels. Antigen binding was not impacted by the presence of this size variant suggesting a non-covalent association of Tyr substituted HHL and LC. The latter complex is not maintained in the denaturing conditions associated with CE-SDS and RPLC-MS. Its existence could, nevertheless, be confirmed by native SEC-MS which preserves non-covalent protein interactions during separation and electrospray ionization. Amino acid analysis furthermore demonstrated a depletion of Cys during the fed-batch process indicating that the observed size/sequence variant is not of genetic but rather of metabolic origin. Native SEC-MS showed that supplementing the cell culture medium with Cys halts misincorporation of Tyr and promotes the formation of the desired mAb structure. To the best of our knowledge, Cys-to-Tyr substitutions preventing interchain disulfide bridge formation have not been described earlier. This observation adds to the impressive structural heterogeneity reported to date for mAbs.